The present invention relates to a method of evaluating an optical analysis device and a phantom sample.
Hitherto, an optical analysis device capable of quantitatively observing a state and characteristics of light emitting particles in an aqueous solution having a lower concentration than those dealt with in optical analysis techniques including a statistical process, such as fluorescence correlation spectroscopy (FCS) has been known (for example, International Publication No. WO2011/108369).
This optical analysis device uses an optical system capable of detecting light from a confocal volume in the aqueous solution, as in case of a confocal microscope or a multi-photon excitation microscope. While the position of the confocal volume is moved in the aqueous solution, that is, while the aqueous solution is scanned using the confocal volume, when light emitting particles which are dispersed in the aqueous solution and randomly move are included in the confocal volume, the light emitted from the light emitting particles is detected.
Basically, this optical analysis device measures the concentration of the light emitting particles sufficiently discretely dispersed in the aqueous solution with respect to the size of the confocal volume, and is based on a very plain and simple principle that the pulse sequence of the fluorescence intensity (photon) obtained when the confocal volume scanned at a speed higher than a speed of the diffusion of the particles by Brownian movement of water in the aqueous solution passes through the particle reflects a bell shape which is an intensity distribution of the confocal volume, and this characteristic shape is determined as one particle.
One passing particle is counted as one continuous bell-shaped mountain (peak), and the sum of the number of peaks during a set scanning time is proportional to the concentration of the particles in the aqueous solution. That is, when target particles are previously allowed to exist at a known concentration, the number of peaks during scanning for a set time is measured, and this operation is performed on aqueous solutions having a series of concentrations, the concentration and the number of peaks can be calibrated. As a result, when an aqueous solution having an unknown concentration is subjected to the measurement and the number of peaks thereof is obtained, the concentration can be measured.
When the optical system of the foregoing optical analysis device is evaluated, a plurality of types of aqueous solutions of fluorescent dye molecules having different known concentrations in practice is prepared, and the number of peaks of the fluorescent light obtained when confocal volumes are scanned over a predetermined time in the respective aqueous solutions is associated with the concentration to perform an operation of calibrating the relationship between the number of peaks and the concentration.
At this time, when a state in which the number of peaks obtained with respect to a predetermined concentration is smaller than the number estimated from the optical system, or a state in which the height of the pulse sequence of photons forming the peak is low, i.e., the fluorescent light has a low intensity is confirmed, these indicate that the optical system is not in an accurate state. Here, as elements which determine the state, there are the size of the confocal volume provided in the aqueous solution, the height of the confocal volume from a surface of a transparent bottom plate of the container in which the aqueous solution is held (the position of the focal point), the scanning speed, the sensitivity of the detector, deviation of an optical axis, and the like.